Faustino Mollinedo

Microtubules, microtubule-interfering agents and apoptosis

Microtubules are dynamic polymers that play crucial roles in a large number of cellular functions. Their pivotal role in mitosis makes them a target for the development of anticancer drugs. Microtubule-damaging agents suppress microtubule dynamics, leading to disruption of the mitotic spindle in dividing cells, cell cycle arrest at M phase, and late apoptosis. A better understanding of the processes coupling microtubule damage to the onset of apoptosis will reveal sites of potential intervention in cancer chemotherapy. Inhibition of microtubule dynamics induces persistent modification of biological processes (M arrest) and signaling pathways (mitotic spindle assembly checkpoint activation, Bcl-2 phosphorylation, c-Jun NH2-terminal kinase activation), which ultimately lead to apoptosis through the accumulation of signals that finally reach the threshold for the onset of apoptosis or through diminishing the threshold for engagement of cell death. Microtubules serve also as scaffolds for signaling molecules that regulate apoptosis, such as Bim and survivin, and their release from microtubules affect the activities of these apoptosis regulators. Thus, sustained modification of signaling routes and changes in the scaffolding properties of microtubules seem to constitute two major processes in the apoptotic response induced by microtubule-interfering agents.

Intracellular Triggering of Fas Aggregation and Recruitment of Apoptotic Molecules into Fas-enriched Rafts in Selective Tumor Cell Apoptosis

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium

Capsaicin is a vanilloid quinone analog that inhibits the plasma membrane electron transport (PMOR) system and induces apoptosis in transformed cells. Using a cytofluorimetric approach we have determined that capsaicin induces a rapid increase of reactive oxygen species (ROS) followed by a subsequent disruption of the transmembrane mitochondrial potential (ΔΨm) and DNA nuclear loss in transformed cell lines and in mitogen activated human T cells. This apoptotic pathway is biochemically different from the typical one induced by either ceramide or edelfosine where, in our system, the ΔΨm dissipation precedes the generation of reactive oxygen species. Neither production of ROS nor apoptosis was found in capsaicin-treated resting T cells where the activity of the PMOR system is minimal when compared with mitogen activated or transformed T cells. Capsaicin also induces Ca2+ mobilization in activated but not in resting T cells. However, preincubation of cells with BAPTA-AM, which chelate cytosolic free calcium, did not prevent ROS generation or apoptosis induced by capsaicin, suggesting that ROS generation in capsaicin treated cells is not a consequence of calcium signaling and that the apoptotic pathway may be separated from the one that mobilizes calcium. Moreover, we present data for the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation. These results provide evidence that the PMOR system may be an interesting target to design antitumoral and anti-inflammatory drugs.

Biological Activities, Mechanisms of Action and Biomedical Prospect of the Antitumor Ether Phospholipid ET-18-OCH3 (Edelfosine), A Proapoptotic Agent in Tumor Cells

The antitumor ether lipid ET-18-OCH(3) (edelfosine) is the prototype of a new class of antineoplastic agents, synthetic analogues of lysophosphatidylcholine, that shows a high metabolic stability, does not interact with DNA and shows a selective apoptotic response in tumor cells, sparing normal cells. Unlike currently used antitumor drugs, ET-18-OCH(3) does not act directly on the formation and function of the replication machinery, and thereby its effects are independent of the proliferative state of target cells. Because of its capacity to modulate cellular regulatory and signaling events, including those failing in cancer cells, like defective apoptosis, ET-18-OCH(3), beyond its putative clinical importance, is an interesting model compound for the development of more selective drugs for cancer therapy. Although ET-18-OCH(3) enhances host defense mechanisms against tumors, its major antitumor action lies in a direct effect on cancer cells, inhibiting phosphatidylcholine biosynthesis and inducing apoptosis in tumor cells. Recent progress has allowed unraveling the molecular mechanism underlying the apoptotic action of ET-18-OCH(3), leading to the notion that ET-18-OCH(3) is selectively incorporated into tumor cells and induces cell death by intracellular activation of the cell death receptor Fas/CD95. This intracellular Fas/CD95 activation is a novel mechanism of action for an antitumor drug and represents a new way to target tumor cells in cancer chemotherapy that can be of interest as a new framework in designing novel antitumor drugs. ET-18-OCH(3) and some analogues are pleiotropic agents that affect additional biomedical important diseases, including parasitic and autoimmune diseases, suggesting new therapeutic indications for these compounds.

Intracellular triggering of Fas, independently of FasL, as a new mechanism of antitumor ether lipid‐induced apoptosis

Antitumor ether lipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET‐18‐OCH3 required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET‐18‐OCH3 does not promote Fas or FasL expression and ET‐18‐OCH3‐induced apoptosis is not inhibited by pre‐incubation with an anti‐Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET‐18‐OCH3‐resistant normal human Fas‐positive fibroblasts do not incorporate ET‐18‐OCH3, but undergo apoptosis upon ET‐18‐OCH3 microinjection. Murine fibroblasts L929 and L929‐Fas, stably transfected with human Fas cDNA, do not incorporate ET‐18‐OCH3 and are resistant to its action when added exogenously. Microinjection of ET‐18‐OCH3 induces apoptosis in L929‐Fas cells, but not in wild‐type L929 cells. Confocal laser scanning microscopy shows that ET‐18‐OCH3 induces Fas clustering and capping during triggering of ET‐18‐OCH3‐induced apoptosis. Microinjection‐induced apoptosis and Fas clustering are specific for the molecular structure of ET‐18‐OCH3. Our data indicate that ET‐18‐OCH3 induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET‐18‐OCH3 on tumors since only cancer cells incorporate sufficient amounts of the drug. Int. J. Cancer 85:674–682, 2000.

Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells.

Neutrophils possess a very short lifespan, dying by apoptosis. HL‐60 cells undergo apoptosis after neutrophil differentiation with dimethyl sulfoxide (DMSO). We have found that the onset of apoptosis in neutrophil‐differentiating HL‐60 cells correlates with the achievement of an apoptosis‐related gene expression pattern similar to that of peripheral blood mature neutrophils. Using reverse transcriptase‐polymerase chain reaction, cloning, and sequencing techniques, we have found that HL‐60 cells express bak, bik, bax, bad, bcl‐2, bcl‐xL , bcl‐w, bfl‐1, fas, and caspases 1–4 and 7–10. After DMSO treatment, bak, bcl‐w, bfl‐1, fas, and caspases 1 and 9 were up‐regulated, whereas bik, bcl‐2, and caspases 2, 3, and 10 were down‐regulated at different degrees, achieving mRNA expression levels that correlated with those detected in peripheral blood neutrophils. Caspase‐2 mRNA and protein expression was drastically reduced after HL‐60 cell differentiation, being absent in both HL‐60‐differentiated neutrophils and mature neutrophils, whereas caspase‐3 and ‐10 mRNA and protein expression were diminished upon HL‐60 cell differentiation until achieving the respective levels found in mature neutrophils. Bak and bfl‐1 mRNA levels were largely increased during DMSO‐induced differentiation of HL‐60 cells, and these genes were the bcl‐2 family members that were expressed most abundantly in mature neutrophils. Bcl‐2 overexpression or caspase inhibition prevented differentiation‐induced apoptosis in HL‐60 cells, but not their differentiation capability. Neutrophil spontaneous apoptosis was also blocked by the caspase inhibitor z‐Asp‐2, 6‐dichlorobenzoyloxymethylketone. Peripheral blood neutrophils expressed bak, bad, bcl‐w, bfl‐1, fas, and caspases 1, 3, 4, and 7–10, but hardly expressed bcl‐2, bcl‐xL , bik, bax, and caspase‐2. These results suggest that the above gene expression changes in neutrophil‐differentiating HL‐60 cells may play a role in the acquisition of the neutrophil apoptotic features. J. Leukoc. Biol. 67:712–724; 2000.

Involvement of mitochondria and caspase-3 in ET-18-OCH3-induced apoptosis of human leukemic cells

The induction of cell death in leukemic HL‐60 cells by the ether lipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET‐18‐OCH3 induced disruption of the mitochondrial transmembrane potential (ΔΨm) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET‐18‐OCH3 also induced caspase‐3 activation in human leukemic cells, as assessed by cleavage of caspase‐3 into the p17 active form and cleavage of the caspase‐3 substrate poly(ADP‐ribose) polymerase (PARP). ET‐18‐OCH3 analogues unable to induce apoptosis failed to disrupt ΔΨm and to activate caspase‐3. ET‐18‐OCH3‐resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase‐3 activation and did not undergo ΔΨm disruption upon ET‐18‐OCH3 incubation. Cyclosporin A partially inhibited ΔΨm dissipation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic cells. Overexpression of bcl‐2 by gene transfer prevented ΔΨm collapse, ROS generation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic T cells. Pretreatment with the caspase inhibitor Z‐Asp‐2,6‐dichlorobenzoyloxymethylketone prevented ET‐18‐OCH3‐induced PARP proteolysis and DNA fragmentation, but not ΔΨm dissipation. ET‐18‐OCH3 did not affect the expression of caspases and bcl‐2‐related genes. ET‐18‐OCH3‐induced apoptosis did not require protein synthesis. Our data indicate that ΔΨm dissipation and caspase‐3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET‐18‐OCH3, and that the sequence of events in the apoptotic action of ET‐18‐OCH3 on human leukemic cells is: ΔΨm disruption, caspase‐3 activation and internucleosomal DNA degradation. Int. J. Cancer 86:208–218, 2000.

Involvement of raft aggregates enriched in Fas/CD95 death-inducing signaling complex in the antileukemic action of edelfosine in Jurkat cells.

Background Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis. Methodology/Principal Findings By a combination of genetic, biochemical, and ultrastructural approaches, we provide here compelling evidence for the involvement of lipid raft aggregates containing recruited Fas/CD95 death receptor, Fas-associated death domain-containing protein (FADD), and procaspase-8 in the induction of apoptosis in human T-cell leukemia Jurkat cells by the antitumor drug edelfosine, the prototype compound of a promising family of synthetic antitumor lipids named as synthetic alkyl-lysophospholipid analogues. Co-immunoprecipitation assays revealed that edelfosine induced the generation of the so-called death-inducing signaling complex (DISC), made up of Fas/CD95, FADD, and procaspase-8, in lipid rafts. Electron microscopy analyses allowed to visualize the formation of raft clusters and their co-localization with DISC components Fas/CD95, FADD, and procaspase-8 following edelfosine treatment of Jurkat cells. Silencing of Fas/CD95 by RNA interference, transfection with a FADD dominant-negative mutant that blocks Fas/CD95 signaling, and specific inhibition of caspase-8 prevented the apoptotic response triggered by edelfosine, hence demonstrating the functional role of DISC in drug-induced apoptosis. By using radioactive labeled edelfosine and a fluorescent analogue, we found that edelfosine accumulated in lipid rafts, forming edelfosine-rich membrane raft clusters in Jurkat leukemic T-cells. Disruption of these membrane raft domains abrogated drug uptake and drug-induced DISC assembly and apoptosis. Thus, edelfosine uptake into lipid rafts was critical for the onset of both co-aggregation of DISC in membrane rafts and subsequent apoptotic cell death. Conclusions/Significance This work shows the involvement of DISC clusters in lipid raft aggregates as a supramolecular and physical entity responsible for the induction of apoptosis in leukemic cells by the antitumor drug edelfosine. Our data set a novel framework and paradigm in leukemia therapy, as well as in death receptor-mediated apoptosis.

ET-18-OCH3 (Edelfosine): A Selective Antitumour Lipid Targeting Apoptosis Through Intracellular Activation of Fas/CD95 Death Receptor

Synthetic ether-linked analogues of phosphatidylcholine and lysophosphatidylcholine, collectively named as antitumour lipids (ATLs), were initially synthesized in the late 60s, but have attracted a renewed interest since the finding that the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), a synthetic analogue of 2-lysophosphatidylcholine considered the ATL prototype, induces a selective apoptotic response in tumour cells, sparing normal cells. Unlike most chemotherapeutic agents currently used, ET-18-OCH3 does not interact with DNA, but act at the cell membrane, and thereby its effects seem to be independent of the proliferative state of target cells. Each part of the molecular structure of ET-18-OCH3 is important for its optimal proapoptotic activity. Recent progress has unveiled the molecular mechanism underlying the apoptotic action of ET-18-OCH3, involving membrane rafts and Fas/CD95 death receptor, and has led to the proposal of a two-step model for the ET-18-OCH3 selective action on cancer cells, namely: a) ET-18-OCH3 uptake into the tumour cell, but not in normal cells; b) intracellular activation of Fas/CD95 through its translocation and capping into membrane rafts. ET-18-OCH3 constitutes the first antitumour drug acting through the intracellular activation of the Fas/CD95 death receptor. Computational docking studies have allowed us to propose a molecular model for the putative interaction of ET-18-OCH3 with the intracellular Fas/CD95 death domain. This novel mechanism of action represents a new way to target tumour cells in cancer chemotherapy and can be of interest as a new framework in designing novel and more selective proapoptotic antitumour drugs.

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding.

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.